[unreadable] Hypoglycemic brain injury is a serious risk of insulin therapy, and this risk is a major impediment to optimal glucose control in diabetic patients. Neuronal death resulting from hypoglycemia is not an inevitable consequence of energy failure, but results instead from a sequence of events initiated by hypoglycemia and glucose re-infusion. It is important to more fully understand the sequence of events in this cell death pathway in order to identify downstream points at which therapeutic interventions can be made to improve neuronal survival and ultimate cognitive function. There has recently been significant progress in this regard, but a cohesive picture of this pathway is still lacking. Studies in this proposal are based on recent salient observations: a) reactive oxygen species (ROS) are generated in neurons at the time of glucose re-infusion (similar to ROS production during reperfusion after ischemia); b) ROS formation leads to DNA damage and PARP-1 activation; c) PARP-1 activation is a major factor mediating hypoglycemic neuronal death; d) zinc chelation blocks hypoglycemia-induced PARP-1 activation and cell death; and e) non-glucose substrates can rescue cells from PARP-1 - mediated cell death. We propose, as a mechanism by which ROS are produced only after glucose re-infusion, that in the absence of glucose there is no substrate for NADH production and consequently no substrate for ROS production by nNOS or NADPH oxidase, and no electron source for the mitochondrial generation of superoxide. Upon glucose re-infusion, ROS formation from each of these sites may occur. The studies proposed here will use cell culture and in vivo models of severe hypoglycemia to identify the major site(s) of ROS formation and the temporal and cause-effect relationships between the events leading to hypoglycemic neuronal death. Proposed studies will also examine therapeutic approaches suggested by these recent observations. A key, novel aspect of these studies is a focus on ROS production initiated during glucose re-infusion. Glucose infusion for severe hypoglycemia is generally initiated by health care providers, and consequently it should in principle be possible to simultaneously administer of adjunctive agents aimed at blocking ROS production or its consequences. We aim to identify effective interventions for this setting. [unreadable] [unreadable]